The short-term objective of this project is to obtain human epidermal stem cells (EpiSCs) in a nearly pure population in order to aid their characterization, to provide evidence for or against a hierarchy of epidermal progenitors, and to study alterations in EpiSC homeostasis that occur due to benign and neoplastic hyperproliferation. The long-term objective is to better understand epidermal differentiation and carcinogenesis enable stem cell-targeted gene therapy, expand populations of EpiSCs for wound-healing applications, and to provide novel therapies for benign and oncogenic cutaneous hyperproliferation. In Aim 1A, for the ongoing goal of quantifying the ability of selection strategies to isolate EpiSCs, and based on 20 years of hematopoietic experience, we have devised a strategy to obtain a nearly pure population of EpiSCs and to assess putative markers in terms of their ability to mark EpiSCs versus short term repopulating (transit amplifying cells, TACs). In Aim 1A i the expression of intracellular stem cell antigens will be used to choose FACS-compatible putative EpiSC markers for quantitative testing in an in vivo transplantation assay. In Aim 1A ii a cocktail of lineage-negative markers will be optimized, akin to that used in hematopoesis, to eliminate cells expressing antigens associated with a proliferative or differentiated state. In Aim 1A iii EpiSCs will be FACS-sorted, using the most effective selection strategy from Aim 1A i along with the lineage-negative cocktail from Aim 1A ii, and the degree of enrichment quantified in the transplantation assay. We will not only study the long-term repopulation ability, but the multipotency, self-renewal, and in vitro growth potential of the most enriched population of EpiSCs that we obtain. In Aim 1B the enrichment of putative TACs in selected keratinocyte populations will be determined and this work will provide evidence for a single epidermal progenitor or a hierarchy of progenitors. In Aim 2 we will examine the hypothesis that oncogenic agents (Bmi-1, Shh, p53) promote EpiSC symmetric self-renewal divisions thereby increasing the stem cell population, while agents that promote benign cutaneous hyperproliferation (IL-1alpha, amphiregulin) will increase asymmetric EpiSC divisions, thus maintaining the EpiSC population and producing an increase in TACs only. Great effort is continuously being exerted to develop sets of EpiSC markers based on a variety of assays that cannot be compared. Our plan is to use available markers in a quantitative and reproducible functional assay of stem cell behavior, to determine the relative ability of existing putative markers to isolate EpiSCs, and most importantly to combine their use strategically, to obtain a nearly pure population of EpiSCs. Our studies will also help in the understanding of EpiSC differentiation and provide insight as to whether a hierarchy of progenitors or a single epidermal progenitor is responsible for production of the epidermis. Studying alterations in EpiSC homeostasis in benign and oncogenic epidermal hyperproliferation will not only inform regarding possible treatment strategies for benign and malignant hyperproliferative cutaneous diseases but also increase our understanding of normal and dysregulated EpiSC homeostasis. PUBLIC HEALTH RELEVANCE: In this proposal we will obtain a nearly pure population of epidermal stem cells and provide evidence for a hierarchy of progenitors or a single epidermal progenitor. Isolation of a nearly pure epidermal stem cell population will be important for regenerative medicine applications (e.g. wounds, burns), targeting stem cells for genetic manipulation, and for more basic studies of epidermal stem cell regulation/differentiation. We will also examine the alterations in stem cell homeostasis associated with benign and neoplastic hyperproliferative stimuli, to better understand dysregulated stem cell homeostasis in benign hyperproliferative and neoplastic cutaneous disease, as well as providing insight into possible therapeutic strategies. Regenerative medicine for wound healing is especially relevant for VA healthcare, as are benign hyperproliferative disorders (psoriasis, chronic dermatitis) and cutaneous neoplasia, all of which are increased in aging veterans.